The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), are leading causes of herpesvirus-associated morbidity and mortality worldwide. An understanding of the molecular mechanisms used by gammaherpesviruses to undergo productive replication, and promote latency and reactivation is key to the rational design of clinical therapeutics. The tegument proteins that are delivered with the virion into a newly infected cell play critical roles during both early and late stages of virus production and are an integral, yet understudied component of chronic herpesvirus infection and associated disease. The role of tegument proteins are difficult to ascertain for EBV and KSHV given their strict host tropism and the lack of robust virus replication in cell culture. Here, we propose to use murine gammaherpesvirus 68 (MHV68) to disect the function of gammaherpesvirus tegument proteins during virus replication and latency. Exciting preliminary results indicate that the ORF75A and ORF75B tegument proteins have distinct roles during early and late stages of virus replication, and these functions impair the ability of the ORF75B null virus to establish latency in the spleens of infected mice. The central hypothesis of this proposal is that the ORF75A and ORF75B tegument proteins of MHV68 have separable roles during virus replication upon de novo productive infection that will impair latency and reactivation in vivo. This proposal describes innovative, high-impact experimental approaches to elucidate the contribution of ORF75 tegument proteins to gammaherpesvirus pathogenesis. In Aim 1, we will discern the role of ORF75A and ORF75B by identifying the defects in virus replication upon conditional ablation of the proteins in the tegument of incoming particles or ablation of newly synthesized protein. In Aim 2, we will use the intraperitoneal route of infection to circumvent lung replication defects in the acute phase upon the intranasal route of inoculation to directly examine the role of ORF75A and ORF75B in the establishment of latency and reactivation from latency in the B cells and macrophages of infected mice. We expect to uncover novel functions that will establish a framework for further mechanistic investigations of ORF75 and provide new insight into the role of tegument proteins as determinants of chronic gammaherpesvirus infection and KSHV- and EBV-associated diseases.